Nori Equine Transferrin ELISA Kit Summary

Alternative names for transferrin: TF, serotransferrin

Alternative names for equine: Horse

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Transferrins are iron-binding blood plasma glycoproteins that control the level of free iron (Fe) in biological fluids. Transferrin is encoded by the TF gene.^[1] transferrin is known as “apotransferrin” when not bound to iron. Transferrins bind iron tightly, but reversibly. Although iron bound to transferrin is less than 0.1% (4 mg) of total body iron, it forms the most vital iron pool with the highest rate of turnover (25 mg/24 h). Transferrin (80 kDa) contains two specific high-affinity Fe(III) binding sites. The affinity of transferrin for Fe(III) is extremely high (association constant is 10²⁰ M⁻¹ at pH 7.4)^[2] but decreases progressively with decreasing pH below neutrality. The pH of the vesicle is reduced by hydrogen ion pumps (H⁺ATPases) to about 5.5, causing transferrin to release its iron ions. When a transferrin protein loaded with iron binds a transferrin receptor on the surface of a cell, it transported into the cell in a vesicle by receptor-mediated endocytosis. The receptor with its ligand bound transferrin is then transported through the endocytic cycle back to the cell surface, ready for another round of iron uptake. Each transferrin molecule can carry two iron ions in the ferric form (Fe³⁺). The liver is the main site of transferrin synthesis but other tissues and organs also produce transferrin. A major source of transferrin secretion in the brain is the choroid plexus in the ventricular system.^[3] The main role of transferrin is to deliver iron from absorption centers in the duodenum and white blood cell macrophages to all tissues. Transferrin plays a key role in areas where erythropoiesis and active cell division occur.^[4] Transferrin is an acute phase protein and is also associated with the innate immune system and the level of transferrin decreases in inflammation,^[5] cancers, and certain diseases.^[6]

References

1. Yang F, et al. (1984). Proc National Academy Sci USA. 81 (9): 2752–6.
2. Aisen P, et al. (1978). The Journal of Biological Chemistry. 253 (6): 1930–7.
3. Moos, T (2002). Danish medical bulletin. 49 (4): 279–301.
4. Macedo MF, de Sousa M (2008). Inflammation & Allergy Drug Targets. 7 (1): 41–52.
5. Ritchie RF, et al. (1999). Journal of Clinical Laboratory Analysis. 13 (6): 273–9.
6. Jain S, et al. (2011). Journal of Pharmacy & Bioallied Sciences. 3 (1): 118–27.